30 healthy, adult New Zealand rabbits, male, weight (3.5 ± 0.3) kg, 12-16 weeks old, were randomly divided into MP group and CS group. Based on different left renal warm ischemia 25min, 35min and 45min, the two groups are further grouped into MP 25min, CS 25 min, MP 35 min, CS 35 min, MP 45 min and CS 45 min, with 5 rabbits in each sub-group.
In vivo model establishment for hypothermic machine perfusion in rabbits
Select two male rabbits (body weight 3.2-3.8kg) each time, and remove their bunk feeders 36-48h before the operation. Make sure that they are fasted 36 to 48hrs but sufficient water should be guaranteed in case of accidental death. The surgical instruments sterilized under high heat and pressure should be placed in the tray spread with a disposable sterile towel prior to the operation. The heating panels and small animal electrotome should be fixed in advance. Then, turn on the air conditioning to maintain the operation room at 25°C/50-70% RH. Finally, switch on the shadowless surgical operating lamp.
(1) Anesthetize and weigh the fasted male rabbits. The rabbits are anesthetized by injecting 1% sodium pentobarbital solution (3ml/kg body weight) in the rabbit’s right ear marginal vein through 20 ml syringe and scalp acupuncture. After the conjunctival reaction disappears or becomes dull, clamp reflex also disappears, the rabbits are all anesthetized with stable breathing and the body tension is reduced. Fix the scalp acupuncture, establish vein channel, and give intravenous injection of 5% glucose solution at 5s/drop. Maintain the rabbit’s basal body temperature (38±0.5°C). (Supplementary photo 1)
(2) Fix the heating plate set at 45.0°C on the operation table. Place the rabbits on the heating plate in supine position with limbs and head fastened to keep breathing unobstructed. (Supplementary photo 2)
(3) Skin preparation: prepare the skin between the xiphoid process and symphysis pubis for ventral midline celiotomy, and make sure sufficient skin of left abdomen and lumbar region are prepared. Prepare the left femoral skin, and paste the electrosurgical dispersive plate on it, meanwhile insert the rectal temperature probe into the anus only after defecation, otherwise the probe will bend, and temperature measurement will be not accurate. (Supplementary photo 3)
(4) Wash hands according to the clinical sterile requirements, and wear the disposable aseptic surgical gowns.
(5) Soak the sterile gauze in povidone iodine, then disinfect the operation skin completely by imbricate method, and repeat this 3 times. (Supplementary photo 4)
(6) Take a disposable aseptic hole-towel to drape the abdominal area, and make sure the surgical area is exposed. Cover with disposable surgical sterile towel, and cut in the middle to get a window hole so that the operation can be revealed from the hole. (Supplementary photo 5)
(7) Connect the electrotome and suction apparatus, then carry out conventional laparotomy with abdominal incision. Gash the skin below the xiphoid process to 3-5cm above the pubic symphysis. If bleeding happens, stop it with pressing gauze and electric coagulation hemostasis, and ligation should be avoided as much as possible. Incise the muscle along the linea alba with the electrotome after hemostasis of skin. Attention should be given to cut muscle layer with slightly raising the muscle to protect the abdominal viscera from injury. More bleeding will happen in the muscle layer, and also liable to hemodiapedesis, electric coagulation hemostasis or incision suture method is often used until hemodiapedesis is completely stopped. (Supplementary photo 6)
(8) Clamping the renal pedicle and abdominal exploration: protect the cut edge with a cotton pad and gauze, push slightly the abdominal organs aside to the right, cover them with gauze, and keep the moisture and temperature with 45.0°C normal saline all the time to prevent intestinal necrosis and dehydration adhesion. Expose the left kidney, and free the left ureter. Blunt dissection of renal vein should be conducted gently to prevent injury and tear. During the blunt dissection of renal artery, whether there is artery malformation or multiple arteries should be determined, and make sure arterial sheath should be isolated well with the length of 1.5-2cm. After finishing the separation, firstly close the ureter by artery clamp, then close renal artery and vein at the same time, start timing from the moment of clipping. Observe the renal color and texture, the kidney should be darkened and harder, while beating of artery should be stopped. Clipping time is 25min/35min/45min. Clip simply the incision skin using tissue forceps to close the abdominal cavity, and maintain the basal body temperature at 38±0.5°C. Draw the blood from the vein of left ear, and carry out blood gas analysis. (Supplementary photo 7)
(9) Release the tissue forceps 1 min before reaching 25min/35min/45min reperfusion, open the abdominal cavity to expose the left kidney, and release two arterial clips the moment reaching 25min/35min/45min. Observe the renal color and texture; the kidney should be seen reddened and softened without petechiae, and pulsation of artery should restore. Be cautious to protect the intestinal tract. Start timing. Simply clip the incision skin using tissue forceps to close the abdominal cavity, and maintain the basal body temperature. Reperfusion time is 1h. (Supplementary photo 8)
(10) Simulation of in vitro renal preservation (CS group)
Before heading to 1h, release the tissue forceps 30min in advance, open the abdominal cavity to expose the left kidney, and be cautious to protect the intestinal tract. At this time, Change 5% glucose solution to hydroxyethyl starch solution I.V. 3 s/drop. Carry out abdominal transverse incision to make cutting edge in parallel with the left renal pedicle, the length is 7-9cm, the transverse incision and the original incision should be shaped like "T", and then stop the bleeding. After completion of hemostasis, give intravenous bolus injection of 0.3ml heparin (1875 IU) via the right marginal ear vein, and then prepare the renal artery puncture device (venous indwelling needle, infusion set and 4°C heparinized citric acid renal protection solution). Isolate the left kidney with the surrounding tissues. Do not cut the ureter and renal pedicle. Perform the ligation to hemostasis, and prevent the infiltration of blood in surrounding tissue. At the moment heading to 1h, clip the renal artery and vein by artery clamp at the same time, and then clip the ureter by the other artery clamp. Then tie two 3-0 mousse line in the renal artery for backup. The left hand pulls the line proximal to the heart, and the right hand punctures the renal artery with head of intravenous indwelling needle. Prepare the crushed ice, 4°C renal preservation solution in citric acid and renal bags. After completion of puncture, open the infusion set, and then prick gently the renal vein with the head of venous indwelling needle after the kidney becomes white, at this time, water column will emit. Observe the perfusing speed preventing the indwelling needle slippage or obstruction. Start countdown for 4h. Firstly ligate the distal end neither too tight nor too loose, to prevent slippage, and then ligate the proximal end. Place the kidney into the bag filled with 4°C citric acid renal preservation solution, tighten the renal bag, and then make sure the bag should not be too tight to prevent the indwelling needle obstruction. Observe the perfusion velocity. Take out the kidney bag from the left body of rabbit, and then perform in vivo renal preservation in the round plate filled with mixture of ice and water. Suture “T” shaped cut, and the kidney was persevered in vivo for 4 hrs. (Supplementary photo 9, 10) (Supplementary video 1)
(11) After completion the 10th step -simulation of in vitro renal preservation (MP group), begin to prepare the drainage tube and lifeport perfusion apparatus. Move the rabbit in proper position so that the left kidney can be pulled out from the transverse incision, but the renal vessels and ureter should not be pulled off. Make sure the streaming velocity does not change, and then place the drainage tube inside, afterwards suture the transverse skin incision to close the left abdomen, which should not influence the streaming velocity. Place the kidney into the sterile plate filled with crushed ice, and bury it with crushed ice. Keep the kidney hypothermia all the time. Install lifeport perfusion apparatus for hypothermia machine perfusion preservation. Afterwards, suture the skin of abdominal median incision, and restore the abdominal temperature. Constantly observe the perfusion flow and the running status of lifeport perfusion apparatus (pressure is 60mmHg, and flow rate should be controlled at 15-20ml/min). At this moment, the hydroxyethyl starch solution can be changed to 5% glucose solution (5s/drop). Monitor the vital signs in the rabbit for 4hrs; make sure there is plenty of ice in the plate. To determine the drainage tube is unobstructed, perform the second blood gas analysis when preserving 2hrs. (Supplementary photo 11) (Supplementary video 2)
(12) Vascular Sutures
Carry out the third blood gas analysis 20 min in advance before heading to 4h, meanwhile prepare 50ml 4°C Lactated Ringer's solution, 5ml sodium bicarbonate, 2ml heparin, 2ml furosemide, 50ml heparin sodium chloride and 50ml 4°Cnormal saline. Open midline incision and the left abdominal transverse incision 5 min in advance (for MP group, lifeport perfusion apparatus needs to be removed to stop pouring). At the same time, change to hydroxyethyl starch solution I.V. 1 s/drop. Fill 50ml 4°C Lactated Ringer's solution in the kidney to rinse away the residual citric acid renal preservation solution. First suture the vein via 10-0 sized suture needle with prolene; then suture the artery after removing the indwelling needle with 10-0 sized suture needle with prolene. The moisture and temperature should be kept in the intestine during the vascular suture, and pay attention to the vital signs of the rabbit. After completion of the vascular suture, carry out leakage test of clipping distal end of renal artery and vein, the procedure is as the followings: open two artery clamps, and determine whether there is bleeding and stenosis in artery and vein. Recover the renal blood supply. At this moment, give intravenous bolus injection of 0.2ml heparin and 0.5ml furosemide via the right marginal ear vein, and 5ml 5% sodium bicarbonate slowly. Observe the vital signs and kidney status of rabbit. Conduct the fourth blood gas analysis. Close the left abdominal transverse incision carefully layer by layer. Prepare the fistula right now. (Supplementary photo 12, 13)
(13) Bladder Stoma. Determine the fistula position, use the purse-string suture, and make sure the bladder should not be strained to injury. At this moment, give intravenous bolus injection of 0.5ml furosemide through the marginal ear vein.
(14) Right Kidney Resection Separate the right renal pedicle and make double ligation; separate the ureter and make double ligation; release the tissues around the kidney, and make ligation for hemostasis. Cut off the renal pedicle and ureter. Resect the right kidney and stop bleeding. At this moment, give intravenous bolus injection of 0.5ml furosemide through the marginal ear vein. (Supplementary photo 14)
(15) Sort and count the apparatus, rinse the abdominal cavity, and close it carefully layer by layer. Then replace hydroxyethyl starch with lactated Ringer's solution I.V. 5 s/drop. Loosen the rabbit from the fastening device, and dry it with blower to make it restore the body temperature as soon as possible. Clean up the experimental apparatus & equipment, clean the perfusion laboratory, and disinfect with UV for backup.
After the operation, monitor the vital signs, urine volume and blood gas analysis of rabbit for 24hrs. Correct the disorder of acid-base balance timely, and supplement the blood volume, which will contribute to the recovery of left kidney function.
Measure the urine volume 24h after operation. Collect the rabbit’s blood into a tube with anticoagulant, and centrifuge 5 min at 2000 rpm to collect the plasma. Content of blood urea nitrogen (BUN) and creatinine (Cr) should be tested with automatic biochemical analyzer; collect another blood into the tube without anticoagulant, and centrifuge 5 min at 2000 rpm to collect the serum, and test the content of TNF-o, IL-6 with ELISA kit; collect tissue to detect the cell apoptosis by TUNEL. (Supplementary photo 15)
Analyze the data using SPSS17.0 statistical software, and the data are normally distributed through normality test. Measurement data are expressed by mean± standard deviation (X±S). Analyze the inter-group data using one-way ANOVA, and apply analysis of variance in a repeated measures design on the intra-group data. α=0.05, p < 0.05 is considered that the difference is statistically significant.